From prior art, the Maxam-Gilbert method and the Sanger method are known as methods for determining the base sequence of a DNA. For instance, in the Maxam-Gilbert method, steps such as the following are required, for example, to determine the base sequence of a DNA.
(1) Fragmenting DNA extracted from cells and integrating a DNA fragment into Escherichia coli, or the like, using a gene recombination technique, or the like.
(2) Culturing Escherichia coli, or the like, to amplify the integrated DNA fragment (cloning).
(3) Lysing Escherichia coli or the like, separating biochemically the integrated DNA fragment, and carrying out of DNA purification with high purity.
(4) Based on the purified DNA fragment, preparing DNA fragments that are different in length by one base each, using the PCR technique. In this step, four species of label are added to the DNA fragments corresponding to the four species of base.
(5) Purifying the reaction product of (4).
(6) Separating by electrophoresis according to the molecular weight of the DNA fragments that are different in length by one base each, and detecting the fluorescence to determine the base sequence of DNA.
Although such method as described above requires cloning, the length of a DNA fragment that can be cloned at once is from several thousands to several tens of thousands base pairs. Consequently, in order to determine, for instance, the base sequence of the total genome for a human, which is approximately three billion base pairs, cloning on the order of at least 100,000 times is necessary.
Thus, with the DNA base sequence determination method that has been used in prior art, there is the problem that considerable effort and time, and enormous costs are necessary in order to determine the base sequence of a DNA. In particular, since the genomic information of higher organisms reach as high as several billions of base pairs, national projects have been necessary, for instance, to determine the base sequence of the total genomic DNA of one human.